United States 

Department of 

Agriculture

Animal and Plant 

Health Inspection 

Service 

Veterinary Services 

Washington, DC 

20250 

April 13, 2012 

VETERINARY SERVICES MEMORANDUM NO. 800.114

TO: VS Leadership Team (VSLT) 

Directors, Center for Veterinary Biologics 

Biologics Licensees, Permittees, and Applicants 

FROM: John R. Clifford /s/ John R. Clifford 

Deputy Administrator 

SUBJECT: Alternative Test Procedure for Tuberculin, PPD Bovis, Intradermic 

I. PURPOSE

The purpose of this memorandum is to inform interested parties that the Center for 

Veterinary Biologics (CVB) is making available an alternate testing protocol to the test 

procedure codified in title 9, Code of Federal Regulations (9 CFR) section113.409(c) for 

Tuberculin-PPD Bovis, Intradermic. Information on obtaining an exemption to use the 

modified test protocol is supplied in this memorandum. 

II. BACKGROUND 

9 CFR 113.409(c) outlines the test procedure for potency testing serials of Tuberculin, 

PPD Bovis, Intradermic (PPD). For this test, 43 guinea pigs are needed: 20 sensitized to 

M. avium, 20 sensitized to M. bovis, and 3 non-sensitized to be used as controls. The 

sensitinogens are prepared from heat-killed mycobacterial cells. A reference and test 

serial of PPD are diluted based on protein content and the dilutions are intradermally 

injected 35 days after the sensitization injection. Each guinea pig receives four injections 

of diluted PPD (i.e., different protein concentrations). 

An alternate testing protocol has demonstrated more reproducibility and ruggedness in 

our laboratory. Modifications to our test method are based on protocols from the World 

Organization for Animal Health (OIE) and the Canadian Food Inspection Agency 

(CFIA). 

The alternate testing protocol uses only 15 guinea pigs, eliminates the M. avium 

sensitized guinea pigs, reduces the number of PPD dilutions for the test, and uses 6 

injections per guinea pig. The dilutions are also distributed amongst the guinea pigs 

differently, so a comparison of the unknown and reference are made within the same 

guinea pig. The skin reactions are evaluated as a percentage of a reference PPD for the 

test interpretation. 

Safeguarding American Agriculture

Federal Relay Service

APHIS is an agency of USDA’s Marketing and Regulatory Programs (Voice/TTY/ASCII/Spanish) 

An Equal Opportunity Provider and Employer 1-800-877-8339 
VETERINARY SERVICES MEMORANDUM 800.114

Page 2 

III. POLICY 

Either the codified test (9 CFR 113.409 and SAM 636) or the modified test procedure 

(BBPRO0002.03 (copy attached)) may be used to test Tuberculin, PPD Bovis, 

Intradermic. The Outline of Production (the Outline) should specify the protocol that will 

be used. The CVB laboratory will use the procedure referenced in the Outline when 

conducting confirmatory testing of serials. 

IV. ACTION 

In order to obtain an exemption to cite the modified protocol in the Outline, a firm should 

test three serials with the method codified in 9 CFR 113.409 (further details supplied in 

SAM 636) and the attached protocol. 

These data should be submitted to your reviewer with the necessary changes to the 

Outline. A Special Outline may be drafted based on the attached protocol. Please ensure 

the data includes the lots of sensitinogen and reference PPD used for the evaluation. 

Authorization to submit samples of the three serials for confirmatory testing will then be 

provided. When satisfactory confirmatory testing is conducted, the alternative protocol 

can be approved for the firm. 

Attachment 
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BBPRO0002.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

Modified Potency Testing Protocol for Purified Protein Derivative (PPD) 

Tuberculins

Date: June 29, 2011

Number: BBPRO0002.03 

Supersedes: BBPRO0002.02, June 2, 2011 

Contact: Janet M. Wilson, (515) 337-7245 

Renee M. Olsen, (515) 337-7467

Approvals: 

/s/Geetha B. Srinivas Date: 08Jul11 

Geetha B. Srinivas, Section Leader 

Bacteriology 

/s/Rebecca L.W. Hyde Date: 08Jul11 

Rebecca L.W. Hyde, Section Leader 

Quality Management 

Center for Veterinary Biologics 

United States Department of Agriculture 

Animal and Plant Health Inspection Service 

P. O. Box 844 

Ames, IA 50010 

INTERNAL USE ONLY

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable. 
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Testing Protocol

BBPRO0002.03

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Modified Potency Testing Protocol for Purified Protein Derivative (PPD) Tuberculins

Table of Contents

1. Introduction

2. Materials

2.1 Instrumentation/equipment

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Selection and handling of test animals

3.3 Preparation of supplies

3.4 Preparation of reagents

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
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Modified Potency Testing Protocol for Purified Protein Derivative (PPD) Tuberculins

1. Introduction

This Testing Protocol (PRO) describes an alternative potency test for evaluation of production 

lots of tuberculin purified protein derivative (PPD). 

Sensitizing inoculums are heat-killed mycobacterial cells suspended in a mixture of mineral oil 

at a concentration of 20 mg/mL (weight/volume). Guinea pigs are injected intramuscularly (IM) 

with this inoculum in order to stimulate an immune response to the tuberculo-proteins which are 

subsequently administered intradermally (ID) when the potency of tuberculin lots are assayed. 

2. Materials

2.1 Instrumentation/equipment

Equivalent instrumentation or equipment may be substituted for any brand name listed 

below. 

2.1.1 Calibrated digital calipers or metric ruler made of clear plastic 

2.1.2 Needles, 20-gauge x 1-inch and 26-gauge x 3/8-inch 

2.1.3 Disposable Luer-locking syringes, 1-mL and 3-mL 

2.1.4 Pipettes, 1-mL, 2-mL, 5-mL and 10-mL 

2.1.5 Polystyrene tubes to dilute the tuberculin serial to 1.0 mg/mL (if needed) 

2.1.6 Glass serum bottles, 10-mL and 20-mL 

2.1.7 Rubber stoppers for serum bottles 

2.1.8 Aluminum seals for serum bottles 

2.1.9 Crimper for applying aluminum seals 

2.1.10 Animal clippers, equipped with a sharpened #40 or #50 blade 

2.1.11 Ear tags for small animals 

2.1.12 Ear tag applicator 

2.1.13 Cage cards 

2.1.14 Calibrated scale suitable for weighing guinea pigs 
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2.1.15 Vivarium specific footwear for animal work 

2.1.16 Scrub pants/top for animal work 

2.1.17 Tape, various colors for identifying each sample 

2.1.18 Personal protective equipment (gloves, eye protection, and/or respirator, if 

allergies present) 

2.1.19 Hotplate 

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. 

2.2.1 International Standard Reference PPD tuberculin, current lot. This 

reference is obtained from the National Institute for Biological Standards and 

Control (NIBSC). 

2.2.2 Mycobacterium bovis sensitizing agent, current lot, for evaluating M. bovis 

tuberculin serials. This reagent is available from the National Veterinary Services 

Laboratories (NVSL). 

2.2.3 Mycobacterium avium sensitizing agent, current lot, for evaluating 

M. avium tuberculin serials. This reagent is available from the NVSL. 

2.2.4 Sterile Saline, 0.85% – National Centers for Animal Health (NCAH) 

Media #30201 

2.2.5 Sterile Mineral Oil 

2.2.6 Phosphate Buffered Saline – NCAH Media #10559 with 0.0005% Tween 

80 

2.2.7 White, non-pregnant female guinea pigs each weighing 500-700 grams. 

Twelve guinea pigs are required for each test serial evaluated and 3 additional 

guinea pigs per serial are held as controls. All guinea pigs must be from same 

source and housed in the same manner. 
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3. Preparation for the Test

3.1 Personnel qualification/training

Technical personnel must have working knowledge of the use of general laboratory 

chemicals, equipment, and glassware and have specific training and experience in the 

safe handling of laboratory animals. They must have experience in the performance of 

this assay. 

3.2 Selection and handling of test animals

3.2.1 Select guinea pigs that are healthy and free of external parasites and have 

an unblemished hair coat. 

3.2.2 Animal care staff will examine the guinea pigs the day they are received, 

and house them according to the current standard operating procedures. 

3.2.3 At the conclusion of the test, the guinea pigs are euthanized unless they 

can be used for other purposes. 

3.3 Preparation of supplies

3.3.1 All glassware must be sterilized prior to use. 

3.3.2 Only sterile supplies must be used (syringes, needles, rubber seals, etc.). 

3.4 Preparation of reagents

3.4.1 Phosphate Buffered Saline - NCAH Media #10559 with 0.0005% Tween 

80 

Sodium chloride 8 g 

Potassium chloride 0.2 g 

Sodium phosphate dibasic 1.15 g 

Potassium phosphate monobasic 0.2 g 

Distilled water QS to 1000 mL 

Autoclave at ≥ 121°C for 20 minutes. When cooled, pH to 7.2 and add 0.5 mL of 

1% solution of Tween 80. 

1% Tween 80 

Tween 80 0.1 mL 

Distilled water QS to 10 mL 
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3.4.2 0.85% Sterile Saline - NCAH Media #30201

Sodium chloride 8.5 g 

Distilled water QS 1000 mL 

Combine and mix well. Autoclave at ≥ 121oC for 20 minutes. 

4. Performance of the Test

The procedure described below is to evaluate one test tuberculin, that is, to compare one test 

tuberculin to the appropriate reference standard. Record all potency test information on the 

current versions of BBTWS0215 and BBTWS0210. 

4.1 Sensitization

4.1.1 Heat and maintain the sensitizing inoculum (2 mg [wet weight] heat-killed 

mycobacterial cells [appropriate species] in 0.1 mL sterile mineral oil) to 

approximately 45°C by placing the vial in a beaker of water on a hot plate. 

4.1.2 Withdraw the warmed sensitinogen out of the bottle using a 16-gauge 

needle, then switch to a 20-gauge, 1-inch needle for sensitinogen inoculation. 

Inject 12 guinea pigs (500-700 g) intramuscularly in the hind leg with 0.1 mL 

inoculum. 

4.1.3 Allocate 3 unsensitized animals to a control group. Inject these guinea 

pigs with 0.1 mL sterile saline (also warmed to 45°C). 

4.2 Guinea Pig Preparation

4.2.1 Thirty-five ± 2 days after sensitization, clip the hair from the abdomen and 

sides of each guinea pig as closely as possible to the skin. 

4.2.2 Allow the guinea pigs to rest in their cages a minimum of 4 hours prior to 

administering tuberculin injections. 

4.3 Intradermal (ID) Inoculation

4.3.1 Dilute the test and standard reference tuberculins in PBS with 0.0005% 

Tween 80 to contain 2, 10, and 50 μg protein/mL (see BBTWS0209). 

4.3.2 Label the bottles containing the various dilutions using different colored 

tape for each sample. 

4.3.3 Insert ear tags in each guinea pig for identification purposes. 
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4.3.4 Restrain the guinea pig on its side. First, inject 0.1 mL intradermally of

each standard tuberculin dilution (4, 5, and 6) into sites A, B, and C on the left 

side of the guinea pig. Then position and restrain the guinea pig on its other side 

to inject 0.1 mL of each test serial dilution (1, 2, and 3) into sites D, E, and F on 

the right side of the guinea pig. The three dilutions are systematically assigned to 

the injection sites (see BBTWS0210). 

4.3.5 Similarly, inject the three dilutions of the standard and the three dilutions 

of the serial intradermally on three unsensitized control guinea pigs. 

4.4 Reading of the skin test

Measure the reactions with calipers 48± 2 hours postinjection. Take two perpendicular 

diameter readings of the area of erythema of each injection site. Record the 

measurements on BBTWS0210. 

5. Interpretation of the Test Results

5.1 The worksheets for data analysis are available upon request. 

5.2 Enter the measurements that have been recorded on BBTWS0210 into 

BBTWS0230. 

5.3 Calculate the average reaction area of each dilution for both the test tuberculin 

and reference standard. Calculate the total area of reaction for each tuberculin. The total 

area of the test tuberculin should be 80-120% of that which is determined for the 

standard. 

6. Report of Test Results

Report results of the test(s) as described by standard operating procedures. 

7. References

CFIA-OLF Standard Operating Procedure SOP #MY-PR041.01 

8. Summary of Revisions

Version .03

4.3.4: This section has been revised for clarification. 
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Modified Potency Testing Protocol for Purified Protein Derivative (PPD) Tuberculins

Version .02

 The title of the document has been updated to reflect more accurate verbiage. 

 2.1.4: Pipettes sizes have been updated to reflect equipment that is used in 

preparation of dilutions. 

 2.1.15: Vivarium specific footwear has been added to reflect the current clothing 

policy to perform animal work. 

 3.2.2: This section has been updated to reflect procedures performed by the Animal 

Resources Unit.